Applicant: Dr. James D. Fessenden is an Instructor at the Boston Biomedical Research Institute (BBRI) for his postdoctoral work, Dr. Fessenden has used cell/molecular biology techniques to determine how structural components of the skeletal muscle Ca -release channel, the ryanodine receptor (RyR), contribute to its function. The successful funding of this application will aid Dr. Fessenden in the transition from a mentored scientist to an independent investigator capable of obtaining funding through his own RO1 application. Training Plan: The training plan will consist of two parts; mentored training on protein chemistry-based techniques to study RyR structure-function and formal coursework to amplify the mentored training and provide instruction on the responsible conduct of research. This training will be supervised by the Sponsor, Dr. Noriaki Ikemoto at the Boston Biomedical Research Institute (BBRI). Dr. Ikemoto, a renowned researcher in the field of RyR structural biology, has pioneered the use of fluorescent probes to study conformational mechanisms of the RyR. In addition, a distinguished Advisory Committee consisting of Dr. Paul Leavis, Dr. Renne Chen Lu and Dr. Sherwin S. Lehrer at the BBRI as well as Dr. Paul Allen at the Harvard Medical School will assist the Candidate in developing new skills in protein chemistry/molecular biology. Environment: The Applicant will be trained primarily at the BBRI, a state-of-the-ar: research facility devoted to the study of muscle proteins using biochemical and biophysical techniques. In addition, the Applicant will have access to the facilities and resources offered by the Allen lab located at the Harvard Medical School. Research Plan: This proposal will examine a key modulator site on the skeletal muscle ryanodine receptor isoform (RyR1). The modulator, 4-chloro-m-cresol (4-CmC), is a clinically relevant RyR activator used to diagnose patjents with the skeletal muscle disorder, malignant hyperthermia (MH). Using molecular biology and biochemical techniques, the binding site for 4-CmC will be localized within the RyR1 primary sequence. In addition, the mechanism of enhanced 4-CmC sensitivity of RyR1 channels containing point mutations that result in MH will be determined using conformationally sensitive fluorescent probes. Finally, molecular distances from the 4-CmC binding site to the high affinity ryanodine binding site on RyR1 will be measured using fluorescence resonance energy transfer (FRET). This study will provide a comprehensive examination of the biophysical characteristics of 4-CmC activation of the RyR while also serving as a point of departure for broader explorations of the RyR using FRET-based techniques.